Retention and function of reduced folate coenzymes in cells is dependent on the formation of poly (Gamma-glutamyl) derivatives of these folates. Deletion of their synthesis by mutation is lethal unless all end products of folate metabolism (thymidine, typoxanthine, Gly, Vet) are supplied to cells. The enzyme responsible for synthesis of polyglutamates, folylpolyglutamate synthetase (FPGS), is thus an attractive target for new antifols. Based on encouraging preliminary studies, a series of new antifolates directed against FPGS which contain ornithine and related amino acids instead of glutamate will be synthesized and investigated. Compounds will be tested as FPGS inhibitors, as well as inhibitors and substrates of other key folate enzymes, and their mechanism examined. Their cytotoxic effects and mechanism on both MTX-sensitive and resistant lymphoid and wild type nonlymphoid human leukemia cell lines will be determined. Activity against the latter type will be of particular interest because patients with nonlymphoid leukemia are intrinsically resistant to MTX. These studies will also determine if there is a correlation between cytotoxicity and FPGS inhibition as measured in cells or with the isolated enzyme. Polygluamate synthesis, turnover, and efflux in folate-depleted and replete human leukemia cell lines are currently being compared using MTX as a model since it also utilizes FPGS. Using this experimental system, we hope to elucidate the role of the intracellular folate pool in regulation of polyglutamate synthesis. This same system will allow us to examine the effects of FPGS inhibitors on polyglutamate synthesis intracellularly in the presence and absence of the folate pool. This system may also uncover interesting interactions between the folate pool and inhibitor which may affect polyglutamate synthesis. FPGS inhibitors will also be useful in basic studies on polyglutamates. For example, for the first time, measurement of degradation of polyglutamates in the absence of continued synthesis will be possible.